Novel phytases and uses thereof

ABSTRACT

The present invention relates to variant phytase enzymes and their use thereof.

BACKGROUND OF THE INVENTION

Phytate is the major but indigestible form of phosphorus found inplant-based feeds. It is considered as an anti-nutritional factor (ANF)that needs to be reduced or removed from cereal-based foods and feeds.Under acidic conditions, phytate interacts with positively chargeddietary proteins leading to the formation of phytate-protein aggregatesand precipitates, which results in a decreased accessibility forproteases, and consequently in inefficient protein digestion. Phytatealso acts as a strong chelating agent that binds different vital metalions in foods and feeds in the small intestine of monogastric organisms,leading to nutritional deficiencies of many important minerals likecalcium, zinc, etc. in animals.

Phytase is a phosphatase that catalyzes the hydrolysis of O—P bonds inphytate and releases inorganic usable phosphorous. Phytase playsversatile roles in agricultural and feeding fields. Ruminant animalssuch as cattle and sheep can utilize the phytate in grains as a sourceof phosphorus since they have bacteria in the gut that producesphytases. Non-ruminants like pigs, poultry, fish, dogs, birds, etc.require extrinsic phytase to liberate inorganic phosphorous. Hence,addition of inorganic phosphorous, a non-renewable and expensivemineral, to feeds for monogastric animals is a common practice, whichincurs heavy costs to the feed industry. Consequently, phytase producedfrom various sources have emerged as one of the most effective andlucrative supplement to these species' diets to enhance the nutritionalvalue of animal feeds and decrease animals' phosphorus excretion thatleads to environmental pollution.

Phytase in feeds can be inactivated by temperature during feedprocessing (pelleting), by the low pH or pepsin in the upper part of thegastrointestinal tract of an animal. Selle and Ravindran laid out thecharacteristics for an ideal feed enzyme, namely; 1) a high specificactivity per unit of protein, 2) good thermostability during feedprocessing, 3) high activity in the typical pH range of the animal gut,4) resistance to gastric proteases, and 5) good stability under ambienttemperatures. (SELLE, P. H. and RAVINDRAN, V. (2007) Microbial phytasein poultry nutrition. Animal Feed Science and Technology 135: 1-41.)

The heat treatment of feeds can involve heat alone or a combination ofboth heat and pressure. The most common form of thermal treatment in themanufacture of poultry feeds is pelleting. The pelleting process firstinvolves the mash feed passing through a conditioner. In the conditionerthe cold feed is exposed to dry steam which is added under pressure.This process helps to improve pellet durability and also increases millthroughputs and reduces energy consumption. Under these conditions,plant cells are crushed, which is favorable for the digestion process inanimals. Nissinen found that moderate conditioning less than 85° C. wasoptimal for broiler performance and high conditioning temperature at 95°C. resulted in poorer body weight gain and feed conversion ratio(NISSINEN, V. (1994) The effects and interactions of enzymes andhydrothermal pre-treatments and their contribution to feeding value.International Milling Flour and Feed, May: 21-22.). Pelleting process at65-85° C. usually result in improving the availability of nutrients dueto the rupture of the cell wall matrix (PICKFORD, J. R. (1992) Effectsof processing on the stability of heat labile nutrients in animal feeds,in: GARNSWORTHY, P. C., HARESIGN, W. & COLE, D. J. A. (Eds) RecentAdvances in Animal Nutrition, pp. 177-192 (Butterworth-Heinemann,Oxford, U.K.) and deactivation of enzyme inhibitors present in cereals(SAUNDERS, R. M. (1975) α-Amylase inhibitors in wheat and other cereals.Cereal Foods World 20: 282-285.). The effect of the damage on thephytase activity due to high pressure appears to be small; it is mainlythe high temperature which results from the high energy input thatinactivates the enzyme. Thus, developing a thermostable Phytase providesan attractive solution for cost-effective processes in the feedindustry.

New applications of phytase in human foods are similarly important asthose in animal feeds because indigestible phytate chelates essentialminerals and contributes to deficiencies of these nutrients inapproximately two to three billion people. The applications of phytasein human health and medicine represent other new exciting areas. Inaddition, phytase has great potentials for industrial applicationsincluding food processing and biofuel production. Thermostable phytase,along with xylanase, have been suggested as effective additives in thepulp and paper industry.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention provides variant phytases and methodsof using them. In one aspect, the invention provides compositionscomprising a variant phytase enzyme comprising at least one amino acidsubstitutions as compared to SEQ ID NO:1, wherein said amino acidsubstitution is at a position number selected from the group consistingof 1, 30, 36, 39, 55, 60, 65, 69, 73, 74, 79, 85, 101, 109, 111, 116,118, 120, 137, 138, 139, 141, 146, 157, 159, 176, 180, 183, 184, 185,186, 189, 233, 245, 255, 276, 282, 288, 291, 295, 297, 311, 315, 341,354, 363, 369, 370, 380, 383, 385 and 402.

In another aspect, the invention provides compositions comprising avariant phytase enzyme comprising at least one amino acid substitutionsas compared to SEQ ID NO:1, wherein said amino acid substitution is at aposition number selected from the group consisting of 1, 30, 36, 39, 55,60, 65, 69, 73, 74, 79, 85, 101, 109, 111, 116, 118, 120, 137, 138, 139,141, 146, 157, 159, 176, 180, 183, 184, 185, 186, 189, 233, 245, 255,276, 282, 288, 291, 295, 297, 311, 315, 341, 354, 363, 369, 370, 380,383, 385 and 402, and wherein said variant phytase enzyme is at least95% identical to SEQ ID NO:1. In additional aspects, the variant phytaseenzyme is at least 96%, 97%, 98% or 99% identical to SEQ ID NO:1,although not SEQ ID NO:1.

In a further aspect, the invention provides compositions comprising avariant phytase enzyme comprising at least one amino acid substitutionsas compared to SEQ ID NO:1, wherein said amino acid substitution is at aposition number selected from the group consisting of 1, 30, 36, 39, 55,60, 65, 69, 73, 74, 79, 85, 101, 109, 111, 116, 118, 120, 137, 138, 139,141, 146, 157, 159, 176, 180, 183, 184, 185, 186, 189, 233, 245, 255,276, 282, 288, 291, 295, 297, 311, 315, 341, 354, 363, 369, 370, 380,383, 385 and 402, wherein said variant phytase enzyme has at least atleast 1.1 fold better activity as compared to SEQ ID NO:1 under acondition selected from the group consisting of thermostability at 58°C., thermostability at 66° C., pH stability at pH 4.5 and pH stabilityat pH 5.5.

In an additional aspect, the invention provides variant phytase enzymeswith one or more amino acid substitutions selected from the groupconsisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D, I55V, H60S, H60Q, R65H,D69N, A73D, A73E, K74D, K74P, K74L, Q79L, Q79R, Q79A, Q79G, Q79F, I85V,A101L, A109D, A109E, A109G, A109F, A109P, T111S, T111D, T111Q, A116Y,A116P, A116R, A116S, T118R, T118S, S120R, N137S, N137P, A138V, A138H,A138D, A138P, N139P, N139A, N139H, T141E, T141G, T141A, T141R, S146R,G157Q, G157N, G157L, G157R, G157A, R159Y, N176K, N180T, N180E, K183R,Q184S, D185N, D185L, E186V, E186A, S189T, G233A, Y255D, T245E, M276V,H282N, H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G,E315S, L341Y, L341V, F354Y, K363A, K363L, N369P, T370P, A380R, A380T,A380P, E383S, R385S, R385V, R385T, E402R, E402T, E402D, E402P and E402N.

In a further aspect, the invention provides variant phytase enzymes withamino acid substitutions at one of said positions, two of saidpositions, three of said positions, four of said positions, five of saidpositions, six of said positions, seven of said positions, eight of saidpositions, nine of said positions, ten of said positions, eleven of saidpositions, twelve of said positions, thirteen of said positions,fourteen of said positions, fifteen of said positions, sixteen of saidpositions, seventeen of said positions, eighteen of said positions,nineteen of said positions or twenty of said positions.

In an additional aspect, the invention provides variant phytase enzymescomprising the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P and at least one further amino acidsubstitution selected from the group consisting of Q1S, Q1V, Q1N, Q30K,A36K, T39D, H60S, H60Q, R65H, D69N, A73D, A73E, K74D, K74P, K74L, Q79L,Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F,A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S,S120R, N137S, N137P, A138V, A138H, A138D, A138P, N139P, N139A, N139H,T141E, T141G, T141A, T141R, S146R, N176K, N180T, N180E, K183R, Q184S,D185N, D185L, E186V, E186A, S189T, G233A, T245E, M276V, H282N, H282P,A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S, L341Y,L341V, K363A, K363L, N369P, T370P, E383S, R385S, R385V, R385T, E402R,E402T, E402D, E402P and E402N.

In a further aspect, the invention provides variant phytase enzymescomprising the amino acid substitutions H60Q/D69N/K74D/S120R/N137P andat least one further amino acid substitution selected from the groupconsisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D, I55V, R65H, A73D, A73E,Q79L, Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G,A109F, A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R,T118S, A138V, A138H, A138D, A138P, N139P, N139A, N139H, T141E, T141G,T141A, T141R, S146R, G157Q, G157N, G157L, G157R, G157A, R159Y, N176K,N180T, N180E, K183R, Q184S, D185N, D185L, E186V, E186A, S189T, G233A,Y255D, T245E, M276V, H282N, H282P, A288E, A288R, A288V, V291I, T295I,V297L, G311S, E315G, E315S, L341Y, L341V, F354Y, K363A, K363L, N369P,T370P, A380R, A380T, A380P, E383S, R385S, R385V, R385T, E402R, E402T,E402D, E402P and E402N.

In an additional aspect, the invention provides variant phytasescomprising the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P and atleast one further amino acid substitution selected from the groupconsisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D, R65H, A73D, A73E, Q79L,Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F,A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S,A138V, A138H, A138D, A138P, N139P, N139A, N139H, T141E, T141G, T141A,T141R, S146R, N176K, N180T, N180E, K183R, Q184S, D185N, D185L, E186V,E186A, S189T, G233A, T245E, M276V, H282N, H282P, A288E, A288R, A288V,V291I, T295I, V297L, G311S, E315G, E315S, L341Y, L341V, K363A, K363L,N369P, T370P, E383S, R385S, R385V, R385T, E402R, E402T, E402D, E402P andE402N.

In a further aspect, the invention provides variant phytases comprisingthe amino acid substitutions N139A/N176K/D185N/E402D and at least onefurther amino acid substitution selected from the group consisting ofQ1S, Q1V, Q1N, Q30K, A36K, T39D, I55V, H60S, H60Q, R65H, D69N, A73D,A73E, K74D, K74P, K74L, Q79L, Q79R, Q79A, Q79G, Q79F, I85V, A101L,A109D, A109D, A109E, A109G, A109F, A109P, T111S, T111D, T111Q, A116Y,A116P, A116R, A116S, T118R, T118S, S120R, N137S, N137P, A138V, A138H,A138D, A138P, T141E, T141G, T141A, T141R, S146R, G157Q, G157N, G157L,G157R, G157A, R159Y, N180T, N180E, K183R, Q184S, E186V, E186A, S189T,G233A, Y255D, T245E, M276V, H282N, H282P, A288E, A288R, A288V, V291I,T295I, V297L, G311S, E315G, E315S, L341Y, L341V, F354Y, K363A, K363L,N369P, T370P, A380R, A380T, A380P, E383S, R385S, R385V and R385T.

In an additional aspect, the invention provides variant phytasescomprising the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P/N139A/N176K/D185N/E402Dand at least one further amino acid substitution selected from the groupconsisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D, R65H, A73D, A73E, Q79L,Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F,A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S,A138V, A138H, A138D, A138P, T141E, T141G, T141A, T141R, S146R, N180T,N180E, K183R, Q184S, E186V, E186A, S189T, G233A, T245E, M276V, H282N,H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S,L341Y, L341V, K363A, K363L, N369P, T370P, E383S, R385S, R385V and R385T.

In a further aspect, the invention provides variant phytase enzymeshaving an amino acid substitution set selected from the group consistingof those depicted in FIGS. 5, 6, 7 and 8.

In an additional aspect, the invention provides compositions of variantphytases further comprising animal feed.

In a further aspect, the invention provides nucleic acids encoding thevariant phytase enzymes of the invention.

In an additional aspect, the invention provides expression vectorscomprising the nucleic acids encoding the variant phytase enzymes of theinvention.

In a further aspect, the invention provides host cells comprising theexpression vectors or the nucleic acids of the invention.

In an additional aspect, the invention provides methods of making avariant phytase enzyme comprising culturing the host cells of theinvention under conditions wherein the variant phytase enzyme isproduced, and recovering the enzyme.

In some aspects, the invention relates to phytase variants havingimproved thermal properties, such as thermostability, heat-stability,steam stability, temperature profile, and/or pelleting stability, withthermostable variant enzymes of particular use in many embodiments.

In additional aspects, the invention relates to phytase variants havingimproved pelleting stability and/or improved acid-stability.

The method of the invention thus relates to phytase variants having animproved pH profile.

The method of the invention thus relates to phytase variants havingimproved protease stability, in particular pepsin stability, found innon-ruminant stomachs.

The method of the invention thus relates to phytase variants havingimproved performance in animal feed (such as an improved release and/ordegradation of phytate).

The invention further relates to polynucleotide comprising nucleotidesequences which encode the phytase variants produced by the method,nucleic acid constructs comprising the polynucleotides operably linkedto one or more control sequences that direct the production of thepolypeptide in an expression host, recombinant expression vectorscomprising such nucleic acid constructs, and recombinant host cellscomprising a nucleic acid construct and/or an expression vector.

In an additional aspect, the invention relates to methods for producingphytase variants as provided comprising (a) cultivating a host cell toproduce a supernatant comprising the phytase; and (b) recovering thephytase.

In a further aspect, the invention relates to methods for improving thenutritional value of an animal feed, by adding a phytase variant of theinvention to the feed, processes for reducing phytate levels in animalmanure by feeding an animal with an effective amount of the feed,methods for the treatment of vegetable proteins, comprising the step ofadding a phytase variant to at least one vegetable protein, and the useof a phytase variant of a composition of the invention.

The invention also provides a method for producing a fermentationproduct such as, e.g., ethanol, beer, wine, comprising fermenting acarbohydrate material in the presence of a phytase variant, a method forproducing ethanol comprising fermenting a carbohydrate material in thepresence of a phytase variant and producing ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleic acid and amino acid sequences of the matureEcPhytase G1P, as well as the amino acid and nucleic acid sequence ofthe endogenous signal sequence. In the parental E. coli strain, the G1Pphytase is produced using an endogenous signal sequence, which isclipped off during expression to form the mature G1P enzyme, as isdepicted in FIG. 9. It should be noted that as for the variant phytases,the phytase can be produced in some organisms using a signal sequence,either the depicted signal sequence or a signal sequence exogenous tothe phytase (e.g. a signal peptide from a different protein or organism,or a synthetic (non-naturally occurring) sequence. That is, depending onthe production host organism, the endogeneous (native to the phytase)signal can be used, or, for example, a signal sequence that is native tothe production host can be recombinantly and operably combined with themature sequence. In some production organism embodiments, the phytase isproduced without the use of a signal sequence. Thus, for production inE. coli, for example, the DNA encoding the signal sequence is linked tothe DNA encoding the mature protein.

FIG. 2 depicts the nucleic acid and amino acid sequences of EcPhytaseG2P. The G2P sequence has the following variants as compared to G1P:I55V/G157Q/R159Y/Y255D/F354Y/A380P. The protein and DNA sequences arefor the mature enzyme.

FIG. 3 depicts the nucleic acid and amino acid sequences of EcPhytaseG3P. In addition to the I55V/G157Q/R159Y/Y255D/F354Y/A380P of G2P theG3P variant also has H60Q/D69N/K74D/S120R/N137P such that the G3P hasthe variantsI55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P relativeto G1P.

FIG. 4 depicts the nucleic acid and amino acid sequences of EcPhytaseG4P. In addition to theI55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P of G3P(inclusive of G2P and the G3P additional variants), the G4P variant hasN139A/N176K/D185N/E402D as well (forming the G4P total set ofI55V/H60Q/D69N/K74D/S120R/N137P/N139A/G157Q/R159Y/N176K/D185N/Y255D/F354Y/A380P/E402D).

FIGS. 5A, 5B, 5C and 5D depict a table showing some of the firstgeneration of variant phytases, and their pH and thermostability.CL0000004 is the G1P parent, whose amino acid sequence is SEQ ID NO:1 asshown in FIG. 1. The majority of the variants are single amino acidvariants, as seen in the “AA Mutations” column, which shows the aminoacid substitution (e.g. from T to E for CL00000605 at position 141) ascompared to G1P. The values of the table were determined as shown inExamples 5 and 6.

FIG. 6 depicts a table showing additional first generation of variantphytases, and their pH and thermostability. CL0000004 is the G1P parent,whose amino acid sequence is SEQ ID NO:1 as shown in FIG. 1. TheCL00000430 is the G2P sequence as depicted in FIG. 2. The values of thetable were determined as shown in Examples 5 and 6.

FIGS. 7A, 7B and 7C depict a table showing second generation of variantphytases, and their pH and thermostability. CL00000430 is the G2Pparent, whose amino acid sequence is SEQ ID NO:5 as shown in FIG. 2. Itshould be noted that the amino acid mutations listed in the table arerelative to the G2P, rather than the G1P. That is, in addition to theamino acid mutations of the Figure, all of the variant phytases listedin FIGS. 7A, 7B and 7C also contain the variantsI55V/G157Q/R159Y/Y255D/F354Y/A380P, which are the variants of G2P. Thusthe G3P variant (CL00005023) has I55V/G157Q/R159Y/Y255D/F354Y/A380P inaddition to H60Q/D69N/K74D/S120R/N137P such that the G3P has thevariants I55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380Prelative to G1P. The values of the table were determined as shown inExamples 5 and 6.

FIG. 8 depicts a table showing a third generation of variant phytases,and their pH and thermostability. CL00005023 is the G3P, and theadditional mutations listed in the table are relative to the G3Pvariant, rather than the G1P or G2P. That is, in addition to the aminoacid mutations of the Figure, all of the variant phytases listed in FIG.8 also contain the variantsI55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P relativeto G1P. Thus the G4P total set of variants isI55V/H60Q/D69N/K74D/S120R/N137P/N139A/G157Q/R159Y/N176K/D185N/Y255D/F354Y/A380P/E402D.The values of the table were determined as shown in Examples 5 and 6.

FIGS. 9A and 9B depict the alignment of the G1P (wild type), G2P, G3Pand G4P variant phytases. The signal sequence, containing the first 22amino acids, is double underlined. The catalytic domain is bolded andunderlined, with the catalytic residues in large italic font and thesubstrate binding residues in large bolded font. Note that the number ofFIG. 9 is inclusive of the signal peptide, which is not the numbering ofthe variant positions outlined herein; that is, the variant positionsherein count the glutamine (Q) residue as position 1 of the matureprotein. Thus, the catalytic domain is amino acids 29-374 in the figurebut amino acids 7 to 352 in the mature protein. Similarly, the H39 andD326 catalytic residues of the figure are H17 and D304 in maturenumbering, and the substrate binding residues are R16, R92 and R267.

FIGS. 10A and 10B show the thermal challenge of selected sequences(including G1P, G2P and G3P). The thermal challenge was a 5 minutechallenge at the indicated temperature as discussed in Example 7.

FIGS. 11A and 11B depict a variant table showing some preferred variantsin some embodiments of the invention. As described herein, these may becombined in any combination, and with variant sets as outlined herein.

FIG. 12 shows the thermal challenge of selected sequences (includingG1P, G2P, G3P and G4P). The thermal challenge was a 5-minute challengeat the indicated temperature as discussed in Example 7. % ResidualActivity is calculated as [(activity of variant at anytemperature)/(activity of variant at 63.0° C.)×100%].

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Phytases decompose phytate (inositol hexakisphosphate (IP6) or phyticacid when in the salt form), which is the primary storage of phosphatein plants. Monogastric animals such as swine, poultry and fish (as wellas humans) cannot digest phytate, leading to phosphorus excretion in themanure, which poses an environmental concern in agricultural areas. Inaddition, the phytate can lead to aggregation of proteins, reducingprotein availability, as well as chelate minerals and trace elements,further reducing the available nutrients for the animals.

The addition of phytase to animal feed was introduced several decadesago and can reduce phosphorus excretion by up to 50% while also allowingthe animal better access to the available nutrients. However, under theconditions which are used in the processing of many foods, includingboth animal feeds made from plant sources as well as foods for humans(cereals, etc.) such as higher temperatures and different pHs, many wildtype phytases are not very stable, leading to inefficient conversion ofthe phytate and/or the cost prohibitive addition of more enzyme.Similarly, other uses for phytase such as in the production of biofuelsalso can include higher temperatures and/or different pHs. Accordingly,it is an object of the present invention to provide variant phytaseswith improved properties, including thermostability and otherbiochemical properties as outlined herein, that lead to improvedoutcomes such as less environmental stress due to lowered phosphorusexcretion, better feed conversion to animal weight and better access tonutrients.

II. Definitions

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionI55V refers to a variant polypeptide, in this case a phytase, in whichthe isoleucine at position 55 is replaced with valine. For clarity, aprotein which has been engineered to change the nucleic acid codingsequence but not change the starting amino acid (for example exchangingCGG (encoding arginine) to CGA (still encoding arginine) to increasehost organism expression levels) is not an “amino acid substitution”;that is, despite the creation of a new gene encoding the same protein,if the protein has the same amino acid at the particular position thatit started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233- or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. In the present case, some embodiments utilize G1P, G2P or G3P asparent polypeptides, with the former being preferred.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide is a wildtype sequence, for example the wild type E. coli phytase designated“G1P” herein. As further discussed below, the protein variant sequenceherein will preferably possess at least about 80% identity with a parentprotein sequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Variant protein can referto the variant protein itself, compositions comprising the proteinvariant, or the DNA sequence that encodes it. Thus, by “variant phytase”herein is meant a novel phytase that has at least one amino acidmodification in the amino acid sequence as compared to a parent phytaseenzyme. As discussed herein, in some cases the parent phytase is asecond or higher generation of variant; that is, as shown in FIG. 6, theG2P phytase has 6 amino acid substitutions as compared to the wild typeG1P parent. However, as shown in FIG. 7, the G3P has 5 amino acidsubstitutions as compared to the G2P parent, but a total of 11 aminoacid substitutions as compared to the G1P. Unless otherwise noted or aswill be obvious from the context, the variant phytases of the inventiongenerally are compared to the wild type G1P sequence. Additionally,unless otherwise noted, the variant phytases of the invention areenzymatically active, that is, there is detectable phytase activityusing the phytase assay described in Example 5, using an assay withouttemperature treatment.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group generally comprisenaturally occurring amino acids and peptide bonds. In addition,polypeptides may include synthetic derivatization of one or more sidechains or termini, glycosylation, PEGylation, circular permutation,cyclization, linkers to other molecules, fusion to proteins or proteindomains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Histidine 82 (also referredto as His82 or H82) is a residue at position 82 in the G1P parentalenzyme.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not found in the parent (e.g. G1P)enzyme.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “position” as used herein is meant a location in the sequence of aprotein. In general, the position number (which is more fully discussedbelow) is relative to the first amino acid of the mature phytasesequence, e.g. excluding the signal peptide.

By “phytase” herein is meant a protein with phytase activity. By“phytase activity” herein is meant that the enzyme catalyzes thehydrolysis of phytate (myo-inositol hexakisphosphate) to (1)myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphatesthereof and (3) inorganic phosphate. Enzymes having detectable activityin the assay outlined below and in Examples 5 are considered phytasesherein.

By “identity” in reference to two sequences herein is meant that thesame amino acid is at the same position considering the alignment. Thedegree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the parent amino acid sequencereferred to in the claims (e.g. for G1P, SEQ ID NO:1) is calculated asthe number of exact matches in an alignment of the two sequences,divided by the length of the “invention sequence,” or the length of theSEQ ID NO:1, whichever is the shortest. The result is expressed inpercent identity as calculated below.

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO:1 is used to determine the corresponding amino acid residuein another phytase of the present invention. The amino acid sequence ofanother phytase is aligned with the mature polypeptide disclosed in SEQID NO:1, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO:1 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherphytase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), EMBOSS EMMA employing ClustalW (1.83 orlater; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), andEMBL-EBI employing Clustal Omega (Sievers and Higgins, 2014, Methods MolBiol. 2014; 1079:105-16), using their respective default parameters.

When the other enzyme has diverged from the polypeptide of SEQ ID NO:1such that traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The standardlyaccepted IUPAC single letter or three letter amino acid abbreviation isemployed.

For an amino acid substitution, the following nomenclature is usedherein: Original amino acid, position, substituted amino acid.Accordingly, the substitution of glutamine at position 441 with prolineis designated as “Gln441Pro” or “Q441P”. Multiple mutations areseparated by forward slash marks (“/”), e.g., “I91L/A133G/Y169W”,representing substitutions at positions 91, 133 and 169, respectively.

1 letter Amino acid Abbreviation abbreviation name Ala A Alanine Arg RArginine Asn N Asparagine Asp D Aspartic acid Cys C Cysteine Gln QGlutamine Glu E Glutamic acid Gly G Glycine His H Histidine Ile IIsoleucine Leu L Leucine Lys K Lysine Met M Methionine Phe FPhenylalanine Pro P Proline Ser S Serine Thr T Threonine Trp WTryptophan Tyr Y Tyrosine Val V Valine

By “isolated” in the context of a phytase herein is meant that thepolypeptide is devoid of other proteins. In a particular embodiment thephytase of the invention is isolated. The term “isolated” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95 to 98% pure, as determined by SDS-PAGE. Inparticular, it is preferred that the polypeptides are in “essentiallypure form”, i.e., that the polypeptide preparation is essentially freeof other polypeptide material with which it is natively associated. Thiscan be accomplished, for example, by preparing the polypeptide by meansof well-known recombinant methods or by classical purification methods.

By “recombinant enzyme” herein is meant that the enzyme is produced byrecombinant techniques and that nucleic acid encoding the variant enzymeof the invention is operably linked to at least one exogeneous (e.g. notnative to the parent phytase) sequence, including, for examples,promoters, terminators, signal sequences, etc., as are more fullyoutlined below.

The term “nucleic acid construct” refers to a nucleic acid molecule,either single-stranded or double-stranded, which is isolated from anaturally occurring gene or is modified to contain segments of nucleicacids in a manner that would not otherwise exist in nature or which issynthetic, and which comprises one or more control sequences.

The term “operably linked” refers to a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directsexpression of the coding sequence.

III. Phytases of the Invention

Accordingly, the present invention provides variant phytases withimproved activity that can be used in a variety of applications,including animal and human nutritional and feed products and theproduction of biofuels such as bioethanol.

In general, the variant phytases of the invention have modified,improved biochemical properties as compared to the wild type parentalphytase, “EcPhytase G1P” or “G1P” (e.g. “generation 1 parent”), SEQ IDNO:1 herein, as shown in FIG. 1. The biochemical properties of thevariant phytases that can be improved herein include, but are notlimited to, pH activity, pH stability, thermostability, specificactivity, formulation stability (including liquid, solid and pellets),performance in animals and/or animal feed and protease stability.

The variant phytases of the invention have one or more improvedproperties as compared to G1P. By “improved” herein is meant a desirablechange of at least one biochemical property. “Improved function” can bemeasured as a percentage increase or decrease of a particular activity,or as a “fold” change, with increases of desirable properties (e.g. pHstability, thermostability) or decreases of undesirable properties (e.g.protease sensitivity). That is, a variant phytase may have a 10%increase in thermostability or a 10% decrease in protease sensitivity,as compared to G1P. Alternatively, a variant phytase may have a 2-foldincrease in pH stability or a 3-fold decrease in protease sensitivity.In general, percentage changes are used to describe changes inbiochemical activity of less than 100%, and fold-changes are used todescribe changes in biochemical activity of greater than 100% (ascompared to the parental enzyme, in many cases G1P). In the presentinvention, percentage changes (usually increases) of biochemicalactivity of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98% and 99% can be accomplished. In the present invention, a “foldincrease” (or decrease) is measured as compared to the starting orparent enzyme. For example, as shown in the Figures, G2P has a 11.64fold increase in temperature tolerance as compared to G1P: this iscalculated by [(activity of variant)/(activity of parent)]. In manyembodiments, the improvement is at least one and a half fold (1.5 fold),2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10fold or higher.

In general, improvements are measured as compared to the G1P enzymeusing a phytase activity assay, under conditions that challenge thevariant phytase against the G1P enzyme.

A. Phytase Assay

The basic phytase assay is run as shown in Example 5 and as follows:after challenge under the appropriate conditions of temperature, pH,etc., the sample is added to a 0.1 M solution of sodium acetatecontaining 2 mM of sodium phytate substrate (C₆H₆Na₁₂O₂₄P₆, FW: 923.81)at pH 4.5 and pH 5.5. The reaction is incubated at 24° C., 150 rpm for30 minutes. The reaction is quenched with half the solution volume of 5%w/v trichloroacetic acid. A sample volume of fresh of coloring reagentis added, which is made by mixing four volumes of 2.5% ammoniummolybdate solution in 5.5% sulfuric acid and one volume of 2.7% ferroussulfate solution. The sample is shaken for 30 seconds and thencentrifuged at 4000 rpm for 2 minutes. A volume of supernatant isdiluted with an equivalent volume of water and absorbance read at 700nm. In some cases, it is useful to use “Phytase Units”, or PU, definedas the amount of phytase required to liberate 1 μmol of inorganicphosphate per minute. The enzyme may be a purified sample, afermentation sample, or a raw sample.

The variant phytases of the invention can have an improvement one ormore of a number of biochemical properties, including, but not limitedto, pH activity, pH stability, thermostability, specific activity,formulation stability (including liquid, solid and pellets), performancein animals and/or animal feed and/or protease stability.

B. Thermostability

In many embodiments, the variant phytases of the invention haveincreased thermostability, particularly under the conditions used toproduce animal feeds, for example, which frequently use hightemperatures during the pelleting process for periods of time thattraditionally inactivate wild type phytases. “Thermostability” in thiscontext means that the variant enzymes are more stable than the parentphytase (e.g. G1P) under the same thermal challenge conditions, that is,the activity of the variant is higher than that of the G1P underidentical conditions (generally using the phytase assay as outlinedherein and as shown in Example 6).

In one embodiment, the variant phytases are more stable than the parentphytase when exposed to temperatures of 40° C., 45° C., 50° C., 55° C.,58° C., 60° C., 65° C., 66° C., 70° C., 75° C., 80° C. and/or 85° C. fora period of time, generally ranging from about 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 minutes or longer, depending on the ultimate conditions for theuse of the variant phytase, with some embodiments utilizing thermalchallenge times of 5 minutes to 10 minutes, 5 minutes to 15 minutes, 5minutes to 60 minutes, 10 minutes to 60 minutes all finding use in thepresent invention. In some embodiments, a challenge of 85° C. and 5minutes is used.

Accordingly, in some embodiments the variant phytases have increasedthermostability as compared to a parent phytase, particularly G1P, forat least 5 minutes at 50° C., at least 5-10 minutes at 55° C., at least5-10 minutes at 58° C., at least 5-10 minutes at 60° C., at least 5-10minutes at 66° C. and in some embodiments at least 5-10 minutes at 70°C.

In addition, pH can be a consideration for thermostability as well.Accordingly, in some embodiments the variant phytases have increasedthermostability as compared to a parent phytase for at least 5 minutesat 58° C. at pH 5.5, at least 5 minutes at 58° C. at pH 4.5, for atleast 5 minutes at 66° C. at 4.5 or at least 5 minutes at 66° C. at pH5.5.

Accordingly, as shown in FIGS. 5, 6, 7 and 8, a number of variantphytases of the invention exhibit increased thermostability.

C. pH Stability

In many embodiments, the variant phytases of the invention haveincreased pH stability at lower pHs, to address the lower pH of thestomach and gastrointestinal tract of non-ruminant animals. That is,many phytases have pH profiles that are suboptimal for the lowered pHenvironment where the activity is desired in the animal “Increased pHstability” in this context means that the variant enzymes are morestable than the parent phytase (e.g. G1P) under the same pH challengeconditions, that is, the activity of the variant is higher than that ofthe G1P under identical conditions (generally using the phytase assay asoutlined herein and as shown in Example 6).

Accordingly, in some embodiments the variant phytases have increased pHstability as compared to a parent phytase, particularly G1P, for atleast 5 minutes at around pH 4.5 and at least 5 minutes at around pH5.5.

D. Specific Activity Assays

In some embodiments, the variant phytases of the invention haveincreased specific activity as compared to a parent phytase,particularly G1P. By “specific activity” herein is meant the activityper amount of enzyme, generally determined by dividing the enzymaticactivity of a sample (sometimes measure in “phytase units” as discussedherein) by the amount of phytase enzyme, generally determined as isknown in the art.

E. Protease Susceptibility

In some embodiments, the variant phytases of the invention are lesssusceptible to protease degradation than the parent enzyme underidentical conditions. In some cases, protease degradation during theproduction of variant phytases in a production host organism by proteaseenzymes produced by the host organism can be a problem, thus resultingin lower yield of active enzyme. This is generally determined as isknown in the art, for example by allowing proteolytic degradation andthen doing N-terminal sequencing on the resulting fragments to determinethe cleavage site(s). In some cases, depending on the variant and thehost production organism, there may not be significant proteolyticdegradation.

As needed, as will be appreciated by those in the art, the specificmutations that can be made will depend on the endogenous proteases thatthe host organism produces, and also generally occurs in surface exposedloop structures or turns that are therefore accessible to proteases. Forexample, production of phytases in A. niger fungal production organismscan lead to proteolytic degradation; see Wyss et al., Appl. And Environ.Microbiol. February 1999:359-366, hereby incorporated by reference inits entirety.

IV. Phytases

Accordingly, the present invention provides variant phytases with one ormore improved properties as compared to the wild type G1P sequence,wherein the phytase is not G1P (SEQ ID NO:1).

In some embodiments, the variant phytases of the invention have at least87% identity to G1P, with enzymes having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% and 99% identity (but less than 100% identity)also finding use in the present invention. Accordingly, some embodimentsprovide variant phytases with from 90% to 99% identity to G1P (SEQ IDNO:1), with other embodiments providing 95% to 99% identity, with theproviso that the phytase is not G1P (SEQ ID NO:1).

In some embodiments, the variant phytases of the invention have at least87% identity to G2P, with enzymes having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% and 99% identity also finding use in the presentinvention, with the proviso that the phytase is not G1P (SEQ ID NO:1).

In some embodiments, the variant phytases of the invention have theamino acid substitutions I55V/G157Q/R159Y/Y255D/F354Y/A380P and are atleast 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5.

In some embodiments, the variant phytases of the invention have at least87% identity to G3P, with enzymes having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% and 99% identity also finding use in the presentinvention, with the proviso that the phytase is not G1P (SEQ ID NO:1).

In some embodiments, the variant phytases of the invention have theamino acid substitutionsI55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P and are atleast 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7.

In some embodiments, the variant phytases of the invention have theamino acid substitutions H60Q/D69N/K74D/S120R/N137P and are at least95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, SEQ ID NO:1 and/orSEQ ID NO:7.

In some embodiments, the variant phytases of the invention have at least87% identity to G4P, with enzymes having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% and 99% identity also finding use in the presentinvention, with the proviso that the phytase is not G1P (SEQ ID NO:1).

In some embodiments, the variant phytases of the invention have theamino acid substitutions N139A/N176K/D185N/E402D and are at least 95%,96%, 97%, 98% or 99% identical to SEQ ID NO:7, SEQ ID NO:1 and/or SEQ IDNO:9.

In some embodiments, the variant phytases of the invention have theamino acid substitutionsI55V/H60Q/D69N/K74D/S120R/N137P/N139A/G157Q/R159Y/N176K/D185N/Y255D/F354Y/A380P/E402Dand are at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9.

V. Specific Variant Phytases

Accordingly, the present invention provides a number of specific variantphytases with improved activity, specifically thermal stability and/orpH stability, and in particular thermal stability at particular pHs andtemperature as outlined herein.

In some embodiments, the variant phytase has one or more amino acidsubstitutions at a position (relative to G1P) selected from the groupconsisting of 1, 30, 36, 39, 55, 60, 65, 69, 73, 74, 79, 85, 101, 109,111, 116, 118, 120, 137, 138, 139, 141, 146, 157, 159, 176, 180, 183,184, 185, 186, 189, 233, 245, 255, 276, 282, 288, 291, 295, 297, 311,315, 341, 354, 363, 369, 370, 380, 383, 385 and 402.

In some embodiments, the variant phytase has one or more amino acidsubstitutions selected from the group consisting of Q1S, Q1V, Q1N, Q30K,A36K, T39D, I55V, H60S, H60Q, R65H, D69N, A73D, A73E, K74D, K74P, K74L,Q79L, Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G,A109F, A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R,T118S, S120R, N137S, N137P, A138V, A138H, A138D, A138P, N139P, N139A,N139H, T141E, T141G, T141A, T141R, S146R, G157Q, G157N, G157L, G157R,G157A, R159Y, N176K, N180T, N180E, K183R, Q184S, D185N, D185L, E186V,E186A, S189T, G233A, Y255D, T245E, Y255D, M276V, H282N, H282P, A288E,A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S, L341Y, L341V,F354Y, K363A, K363L, N369P, T370P, A380R, A380T, A380P, E383S, R385S,R385V, R385T, E402R, E402T, E402D, E402P and E402N.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamine at position 1 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from Q1S, Q1Vand Q1N.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamine at position 30 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is Q30K.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 36 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophan,valine and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is A36K.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 39 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan,valine and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is T39D.

In some embodiments, the variant phytase has an amino acid substitutionof the isoleucine at position 55 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, threonine, leucine, methionine, phenylalanine, tryptophan,valine and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is I55V.

In some embodiments, the variant phytase has an amino acid substitutionof the histidine at position 60 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from H60Q andH60S.

In some embodiments, the variant phytase has an amino acid substitutionof the aspartic acid at position 65 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is R65H.

In some embodiments, the variant phytase has an amino acid substitutionof the aspartic acid at position 69 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is D69N.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 73 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from A73D andA73E.

In some embodiments, the variant phytase has an amino acid substitutionof the lysine at position 74 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, alanine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from K74D,K74L and K74P.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamine at position 79 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from Q79L,Q79A, Q79G, Q79R and Q79F.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 85 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is I85V.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 101 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is A101L.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 109 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from A109D,A109E, A109F, A109P, A109G.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 111 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from T111S,T111D and T111Q.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 116 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from A116Y,A116P, A116R and A116S.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 118 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from T118S andT118R.

In some embodiments, the variant phytase has an amino acid substitutionof the serine at position 120 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely threonine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from S120R.

In some embodiments, the variant phytase has an amino acid substitutionof the asparagine at position 137 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, glutamine, lysine, arginine, histidine,glutamic acid, aspartic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is selected from N137P andN137S.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 138 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from A138V,A138H, A138P and A138D.

In some embodiments, the variant phytase has an amino acid substitutionof the asparagine at position 139 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, glutamine, lysine, arginine, histidine,glutamic acid, aspartic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is selected from N139P, N139Aand N139H.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 141 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from E(T141E), G (T141G), A (T141A), R (T141R).

In some embodiments, the variant phytase has an amino acid substitutionof the serine at position 146 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely threonine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from R(S146R).

In some embodiments, the variant phytase has an amino acid substitutionof the glycine at position 157 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from G157Q,G157N, G157L, G157R, G157A.

In some embodiments, the variant phytase has an amino acid substitutionof the arginine at position 159 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, glutamine, lysine, asparagine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is R159Y.

In some embodiments, the variant phytase has an amino acid substitutionof the asparagine at position 176 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, glutamine, lysine, arginine, histidine,glutamic acid, aspartic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is N176K.

In some embodiments, the variant phytase has an amino acid substitutionof the asparagine at position 180 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, glutamine, lysine, arginine, histidine,glutamic acid, aspartic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is selected from N180T andN180E.

In some embodiments, the variant phytase has an amino acid substitutionof the lysine at position 183 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, alanine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is K183R.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamine at position 184 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is Q184S.

In some embodiments, the variant phytase has an amino acid substitutionof the aspartic acid at position 185 of SEQ ID NO:1. In someembodiments, the substitution is with any other of the 19 naturallyoccurring amino acids, namely serine, threonine, asparagine, glutamine,lysine, arginine, histidine, glutamic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from D185L andD185N.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamic acid at position 186 of SEQ ID NO:1. In someembodiments, the substitution is with any other of the 19 naturallyoccurring amino acids, namely serine, threonine, asparagine, glutamine,lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from E186V andE186A.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 189 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is S189T.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 233 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is G233A.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 245 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is T245E.

In some embodiments, the variant phytase has an amino acid substitutionof the tyrosine at position 255 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, alanine, isoleucine, leucine, phenylalanine, tryptophan valineand methionine, with some embodiments not utilizing cysteine (due topossible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is Y255D.

In some embodiments, the variant phytase has an amino acid substitutionof the methionine at position 276 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, alanine, isoleucine, leucine, phenylalanine, tryptophan valineand tyrosine, with some embodiments not utilizing cysteine (due topossible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is M276V.

In some embodiments, the variant phytase has an amino acid substitutionof the methionine at position 282 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, alanine, isoleucine, leucine, phenylalanine, tryptophan valineand tyrosine, with some embodiments not utilizing cysteine (due topossible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from H282N andH282P.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 288 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from A288E,A288R and A288V.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 291 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is V291I.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 295 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is T295I.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 297 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from V297L.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 311 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is G311S.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamic acid at position 315 of SEQ ID NO:1. In someembodiments, the substitution is with any other of the 19 naturallyoccurring amino acids, namely serine, threonine, asparagine, glutamine,lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from E315G andE315S.

In some embodiments, the variant phytase has an amino acid substitutionof the leucine at position 341 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, alanine, isoleucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from L341Y andL341V.

In some embodiments, the variant phytase has an amino acid substitutionof the phenylalanine at position 354 of SEQ ID NO:1. In someembodiments, the substitution is with any other of the 19 naturallyoccurring amino acids, namely serine, threonine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, alanine, isoleucine, leucine, methionine, asparagine,tryptophan valine and tyrosine, with some embodiments not utilizingcysteine (due to possible disulfide formation) or proline (due to stericeffects). In some embodiments, the amino acid substitution is F354Y.

In some embodiments, the variant phytase has an amino acid substitutionof the lysine at position 363 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from K363A andK363L.

In some embodiments, the variant phytase has an amino acid substitutionof the asparagine at position 369 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, glutamine, lysine, arginine, histidine,glutamic acid, aspartic acid, cysteine, glycine, proline, alanine,isoleucine, leucine, methionine, phenylalanine, tryptophan valine andtyrosine, with some embodiments not utilizing cysteine (due to possibledisulfide formation) or proline (due to steric effects). In someembodiments, the amino acid substitution is N369P.

In some embodiments, the variant phytase has an amino acid substitutionof the threonine at position 370 of SEQ ID NO:1. In some embodiments,the substitution is with any other of the 19 naturally occurring aminoacids, namely serine, asparagine, glutamine, lysine, arginine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is T370P.

In some embodiments, the variant phytase has an amino acid substitutionof the alanine at position 380 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,arginine, histidine, glutamic acid, aspartic acid, cysteine, glycine,proline, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from A380R,A380T and A380P.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamic acid at position 383 of SEQ ID NO:1. In someembodiments, the substitution is with any other of the 19 naturallyoccurring amino acids, namely serine, threonine, asparagine, glutamine,lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is E383S.

In some embodiments, the variant phytase has an amino acid substitutionof the arginine at position 385 of SEQ ID NO:1. In some embodiments, thesubstitution is with any other of the 19 naturally occurring aminoacids, namely serine, threonine, asparagine, glutamine, lysine,histidine, glutamic acid, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from R385V,R385T and R385S.

In some embodiments, the variant phytase has an amino acid substitutionof the glutamic acid at position 402 of SEQ ID NO:1. In someembodiments, the substitution is with any other of the 19 naturallyoccurring amino acids, namely serine, threonine, asparagine, glutamine,lysine, arginine, histidine, aspartic acid, cysteine, glycine, proline,alanine, isoleucine, leucine, methionine, phenylalanine, tryptophanvaline and tyrosine, with some embodiments not utilizing cysteine (dueto possible disulfide formation) or proline (due to steric effects). Insome embodiments, the amino acid substitution is selected from E402D,E402P, E402N, E402R and E402T.

In some embodiments the variant phytase comprises the G2P variantsI55V/G157Q/R159Y/Y255D/F354Y/A380P and a further amino acid substitutionselected from the group consisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D,H60S, H60Q, R65H, D69N, A73D, A73E, K74D, K74P, K74L, Q79L, Q79R, Q79A,Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F, A109P,T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S, S120R,N137S, N137P, A138V, A138H, A138D, A138P, N139P, N139A, N139H, T141E,T141G, T141A, T141R, S146R, N176K, N180T, N180E, K183R, Q184S, D185N,D185L, E186V, E186A, S189T, G233A, T245E, M276V, H282N, H282P, A288E,A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S, L341Y, L341V,K363A, K363L, N369P, T370P, E383S, R385S, R385V, R385T, E402R, E402T,E402D, E402P and E402N.

In some embodiments, the variant phytase comprises the G3P variantsH60Q/D69N/K74D/S120R/N137P, and at least one of the additional singleamino acid variants outlined above, including, but not limited to, Q1S,Q1V, Q1N, Q30K, A36K, T39D, I55V, R65H, A73D, A73E, Q79L, Q79R, Q79A,Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F, A109P,T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S, A138V,A138H, A138D, A138P, N139P, N139A, N139H, T141E, T141G, T141A, T141R,S146R, G157Q, G157N, G157L, G157R, G157A, R159Y, N176K, N180T, N180E,K183R, Q184S, D185N, D185L, E186V, E186A, S189T, G233A, Y255D, T245E,M276V, H282N, H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S,E315G, E315S, L341Y, L341V, F354Y, K363A, K363L, N369P, T370P, A380R,A380T, A380P, E383S, R385S, R385V, R385T, E402R, E402T, E402D, E402P andE402N.

In some embodiments, the variant phytase comprises the G2P and G3Pvariants I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P,and at least one of the additional single amino acid variants outlinedabove, including, but not limited to, Q1S, Q1V, Q1N, Q30K, A36K, T39D,R65H, A73D, A73E, Q79L, Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D,A109D, A109E, A109G, A109F, A109P, T111S, T111D, T111Q, A116Y, A116P,A116R, A116S, T118R, T118S, A138V, A138H, A138D, A138P, N139P, N139A,N139H, T141E, T141G, T141A, T141R, S146R, N176K, N180T, N180E, K183R,Q184S, D185N, D185L, E186V, E186A, S189T, G233A, T245E, M276V, H282N,H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S,L341Y, L341V, K363A, K363L, N369P, T370P, E383S, R385S, R385V, R385T,E402R, E402T, E402D, E402P and E402N.

In some embodiments, the variant phytase comprises the G4P variantsN139A/N176K/D185N/E402D, and at least one of the additional single aminoacid variants outlined above, including, but not limited to, Q1S, Q1V,Q1N, Q30K, A36K, T39D, I55V, H60S, H60Q, R65H, D69N, A73D, A73E, K74D,K74P, K74L, Q79L, Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D,A109E, A109G, A109F, A109P, T111S, T111D, T111Q, A116Y, A116P, A116R,A116S, T118R, T118S, S120R, N137S, N137P, A138V, A138H, A138D, A138P,T141E, T141G, T141A, T141R, S146R, G157Q, G157N, G157L, G157R, G157A,R159Y, N180T, N180E, K183R, Q184S, E186V, E186A, S189T, G233A, Y255D,T245E, M276V, H282N, H282P, A288E, A288R, A288V, V291I, T295I, V297L,G311S, E315G, E315S, L341Y, L341V, F354Y, K363A, K363L, N369P, T370P,A380R, A380T, A380P, E383S, R385S, R385V and R385T.

In some embodiments, the variant phytase comprises the G4P variantsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P/N139A/N176K/D185N/E402Dand a further amino acid substitution selected from the group consistingof Q1S, Q1V, Q1N, Q30K, A36K, T39D, R65H, A73D, A73E, Q79L, Q79R, Q79A,Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F, A109P,T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S, A138V,A138H, A138D, A138P, T141E, T141G, T141A, T141R, S146R, N180T, N180E,K183R, Q184S, E186V, E186A, S189T, G233A, T245E, M276V, H282N, H282P,A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S, L341Y,L341V, K363A, K363L, N369P, T370P, E383S, R385S, R385V and R385T.

Some particular embodiments of the present invention are phytasevariants as compared to SEQ ID NO:1 having an amino acid substitutionset selected from the group consisting of N139H/K183R,R159Y/Y255D/V291I/V297L/G311S, I55V/Y255D/G311S/F354Y,G233A/Y255D/V291I, I85V/G157Q/V291I/V297L/G311S/F354Y, A101L/Y255D,I55V/I85V/Y255D/V291I, I55V/F354Y, I55V/I85V/Y255D/V291I/F354Y,R159Y/Y255D/V291I, A101L/R159Y/S189T/T295I/F354Y, Q30K/I85V/Y255D/A380P,G157Q/R159Y, I55V/I85V/5189T/G233A/Y255D/F354Y/A380P,I55V/I85V/5189T/V297L/G311S, F354Y, I55V/I85V/A101L/G157Q/G233A/F354Y,I55V/G157Q/Y255D/V291I/V297L/F354Y, R159Y, I55V, Y255D,I55V/G157Q/R159Y/Y255D/F354Y/A380P, I55V/R159Y/Y255D/V297L/A380P,I55V/I85V/G157Q/G233A/Y255D/V297L/F354Y, I55V/A101L/G157Q/Y255D/V297L,I55V/A101L/G157Q/Y255D/F354Y, I55V/N291I/V297L, andI55V/I85V/A101L/R159Y/S189T/Y255D/F354Y. Of these,I55V/G157Q/R159Y/Y255D/F354Y/A380P is particularly useful in someembodiments.

In some embodiments, the variant phytases of the invention compriseamino acid substitution sets selected from the group consisting ofT39D/K74D/Q157A, T39D/H60Q/K74D/N137P/T141A, K74D,T39D/D69N/N137P/T141E/Q157A, S120R/N137P/A138V, T39D/H60Q, K74D/T141A,K74P, N137P/A138V, H60Q/D69N, T39D/D69N/K74D,H60Q/D69N/K74D/S120R/N137P, D69N/N137P/A138V/T141E,T39D/D69N/K74P/T111D/S120R/T141A, N137P/T141A, N137P/A138V/T141E,T39D/K74D, T39D/H60S/T111D/S120R, T39D/H60S/D69N/S120R/N137S/T141A,H60Q/N137P/A138V/T141A, Q157L, S120R/N137P, H60Q, S120R,S120R/N137S/A138V/Q157L, H60S/K74Y/S120R/A138V, T39D/D69N/S120R/T141A,H60S, T39D/S120R, T39D, H60S/K74D, T39D/T111D, T39D/H60S,T39D/K74D/T141E, K74D/T111D/T141E/Q157N,H60S/K74D/T111D/S120R/T141E/Q157N, T39D/K74D/S120R/T141E, T141E,K74D/S120R/Q157N, K74D/S120R, T111D/S120R/T141E, H60S/R65H,H60S/D69N/T111D/N137P, T39D/N137S/T141A, H60Q/D69N/N137P/A138V,T39D/D69N/K74D/N137P/A138V/T141E, K74D/T111D/T141A, N137S/A138V/T141E,H60Q/K74P/N137S/T141E, D69N/K74P, H60Q/K74P, T39D/T111D/S120R,T39D/H60Q/K74D/T111D/S120R, T39D/D69N, D69N/K74D, andT39D/H60Q/D69N/N137S/A138V.

In some embodiments, the G2P amino acid variant set,I55V/G157Q/R159Y/Y255D/F354Y/A380P, is added to a second set (the “G3Pset”, above) to provide variant phytase enzymes with an amino acidsubstitution sets selected from the group consisting ofI55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D/Q157A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q/K74D/N137P/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/N137P/T141E/Q157A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R/N137P/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137P/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/D69N/N137P/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/K74P/T111D/S120R/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137P/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137P/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60S/T111D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60S/D69N/S120R/N137S/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/N137P/A138V/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/Q157L,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R/N137P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R/N137S/A138V/Q157L,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/K74Y/S120R/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/S120R/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/T111D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60S,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/T111D/T141E/Q157N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/K74D/T111D/S120R/T141E/Q157N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D/S120R/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/S120R/Q157N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T111D/S120R/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/R65H,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/D69N/T111D/N137P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/N137S/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/N137P/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/K74D/N137P/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/T111D/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137S/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/K74P/N137S/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/D69N/K74P/,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/K74P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/T111D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q/K74D/T111D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/D69N/K74D, andI55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q/D69N/N137S/A138V.

Suitable variant phytases of the invention are those listed in SEQ IDNOs: 12 to 171, and those depicted in the Figures.

VI. Nucleic Acids of the Invention

The present invention additional provides nucleic acids encoding thevariant phytases of the invention. As will be appreciated by those inthe art, due to the degeneracy of the genetic code, an extremely largenumber of nucleic acids may be made, all of which encode the variantphytases of the present invention. Thus, having identified a particularamino acid sequence, those skilled in the art could make any number ofdifferent nucleic acids, by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. Thus, providing the amino acid sequence allows the generationof a very large number of different nucleic acid sequences encoding theproteins.

In some embodiments, specific variant phytases are encoded by specificnucleic acid sequences, as are listed in SEQ ID NOs 172-332.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the heterodimericantibodies of the invention. Generally the nucleic acids are operablylinked to any number of regulatory elements (promoters, origin ofreplication, selectable markers, ribosomal binding sites, inducers,etc.). The expression vectors can be extra-chromosomal or integratingvectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with bacteria and fungi finding use in many embodiments.

A. Preparation of Variants

The nucleic acids encoding the variant phytases of the invention can beprepared using any mutagenesis procedure known in the art, such assite-directed mutagenesis and synthetic gene construction as are wellknown in the art.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips. A preferred technique is GenScript®.

i. Regulatory Sequences

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriphytase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellcan be used.

In some embodiments, terminators for filamentous fungal host cells areobtained from the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger phytase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

In some embodiments, terminators for yeast host cells are obtained fromthe genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence can also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence can also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

In some embodiments, leaders for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase andAspergillus nidulans triose phosphate isomerase.

In some embodiments, suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence can also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

In some embodiments, polyadenylation sequences for filamentous fungalhost cells are obtained from the genes for Aspergillus nidulansanthranilate synthase, Aspergillus niger phytase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant glucoamhylase being expressed into the cell'ssecretory pathway. The 5′-end of the coding sequence of thepolynucleotide may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding sequence that encodes the variant glucoamhylase. Alternatively,the 5′-end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. A foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively, aforeign signal peptide coding sequence may simply replace the naturalsignal peptide coding sequence in order to enhance secretion of thevariant glucoamhylase. However, any signal peptide coding sequence thatdirects the expressed variant into the secretory pathway of a host cellmay be used.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger phytase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. A particular signal sequence is shown in FIG. 1, SEQ ID NO:2.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger phytase promoter, Aspergillusoryzae TAKA alpha-amylase promoter, and Aspergillus oryzae phytasepromoter can be used. Other examples of regulatory sequences are thosethat allow for gene amplification. In eukaryotic systems, theseregulatory sequences include the dihydrofolate reductase gene that isamplified in the presence of methotrexate, and the metallothionein genesthat are amplified with heavy metals. In these cases, the polynucleotideencoding the variant would be operably linked with the regulatorysequence.

1. Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector can be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used. Vectors contemplated for use with themethods of the invention include both integrating and non-integratingvectors.

In some embodiments, the vector contains one or more selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Suitable markers for yeast host cells include, but are not limited to,ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for usein a filamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are Aspergillus nidulans orAspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicusbar gene.

In some embodiments, the vector contains an element(s) that permitsintegration of the vector into the host cell's genome or autonomousreplication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector can rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector can containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector can further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication can be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention can beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

2. Codon Optimization

Codon optimization can be employed with any of the variant phytasepolypeptides of the present invention, in order to optimize expressionin the host cell employed. Such methods are well known in the art anddescribed in, for example, WO 2007/142954. In heterologous expressionsystems, optimization steps can improve the ability of the host toproduce the desired variant phytase polypeptides. Protein expression isgoverned by a host of factors including those that affect transcription,mRNA processing, and stability and initiation of translation. Thepolynucleotide optimization steps can include steps to improve theability of the host to produce the foreign protein as well as steps toassist the researcher in efficiently designing expression constructs.Optimization strategies can include, for example, the modification oftranslation initiation regions, alteration of mRNA structural elements,and the use of different codon biases. The following paragraphs discusspotential problems that may result in reduced heterologous proteinexpression and techniques that may overcome these problems,

In some embodiments, reduced heterologous protein expression resultsfrom a rare codon-induced translational pause. A rare codon-inducedtranslational pause includes the presence of codons in thepolynucleotide of interest that are rarely used in the host organism canhave a negative effect on protein translation due to their scarcity inthe available tRNA pool. One method of improving optimal translation inthe host organism includes performing includes performing codonoptimization which can result in rare host codons being modified in thesynthetic polynucleotide sequence.

In some embodiments, reduced heterologous protein expression resultsfrom by alternate translational initiation. Alternate translationalinitiation can include a synthetic polynucleotide sequence inadvertentlycontaining motifs capable of functioning as a ribosome binding site(RBS). These sites can result in initiating translation of a truncatedprotein from a gene-internal site. One method of reducing thepossibility of producing a truncated protein, which can be difficult toremove during purification, includes modifying putative internal RBSsequences from an optimized polynucleotide sequence.

In some embodiments, reduced heterologous protein expression occursthrough repeat-induced polymerase slippage. Repeat-induced polymeraseslippage involves nucleotide sequence repeats that have been shown tocause slippage or stuttering of DNA polymerase which can result inframeshift mutations. Such repeats can also cause slippage of RNApolymerase. In an organism with a high G+C content bias, there can be ahigher degree of repeats composed of G or C nucleotide repeats.Therefore, one method of reducing the possibility of inducing RNApolymerase slippage includes altering extended repeats of G or Cnucleotides.

In some embodiments, reduced heterologous protein expression occursthrough interfering secondary structures. Secondary structures cansequester the RBS sequence or initiation codon and have been correlatedto a reduction in protein expression. Stemloop structures can also beinvolved in transcriptional pausing and attenuation. An optimizedpolynucleotide sequence can contain minimal secondary structures in theRBS and gene coding regions of the nucleotide sequence to allow forimproved transcription and translation.

In some embodiments, restriction sites can effect heterologous proteinexpression. By modifying restriction sites that could interfere withsubsequent sub-cloning of transcription units into host expressionvectors a polynucleotide sequence can be optimized.

Optimizing a DNA sequence can negatively or positively affect geneexpression or protein production. For example, modifying a less-commoncodon with a more common codon may affect the half life of the mRNA oralter its structure by introducing a secondary structure that interfereswith translation of the message. It may therefore be necessary, incertain instances, to alter the optimized message.

AUG or a portion of a gene can be optimized. In some embodiments, thedesired modulation of expression is achieved by optimizing essentiallythe entire gene. In other embodiments, the desired modulation will beachieved by optimizing part but not all of the gene.

The codon usage of any coding sequence can be adjusted to achieve adesired property, for example high levels of expression in a specificcell type. The starting point for such an optimization may be a codingsequence with 100% common codons, or a coding sequence which contains amixture of common and non-common codons.

Two or more candidate sequences that differ in their codon usage can begenerated and tested to determine if they possess the desired property.Candidate sequences can be evaluated by using a computer to search forthe presence of regulatory elements, such as silencers or enhancers, andto search for the presence of regions of coding sequence which could beconverted into such regulatory elements by an alteration in codon usage.Additional criteria can include enrichment for particular nucleotides,e.g., A, C, G or U, codon bias for a particular amino acid, or thepresence or absence of particular mRNA secondary or tertiary structure.Adjustment to the candidate sequence can be made based on a number ofsuch criteria.

Promising candidate sequences are constructed and then evaluatedexperimentally. Multiple candidates may be evaluated independently ofeach other, or the process can be iterative, either by using the mostpromising candidate as a new starting point, or by combining regions oftwo or more candidates to produce a novel hybrid. Further rounds ofmodification and evaluation can be included.

Modifying the codon usage of a candidate sequence can result in thecreation or destruction of either a positive or negative element. Ingeneral, a positive element refers to any element whose alteration orremoval from the candidate sequence could result in a decrease inexpression of the therapeutic protein, or whose creation could result inan increase in expression of a therapeutic protein. For example, apositive element can include an enhancer, a promoter, a downstreampromoter element, a DNA binding site for a positive regulator (e.g., atranscriptional activator), or a sequence responsible for imparting ormodifying an mRNA secondary or tertiary structure. A negative elementrefers to any element whose alteration or removal from the candidatesequence could result in an increase in expression of the therapeuticprotein, or whose creation would result in a decrease in expression ofthe therapeutic protein. A negative element includes a silencer, a DNAbinding site for a negative regulator (e.g., a transcriptionalrepressor), a transcriptional pause site, or a sequence that isresponsible for imparting or modifying an mRNA secondary or tertiarystructure. In general, a negative element arises more frequently than apositive element. Thus, any change in codon usage that results in anincrease in protein expression is more likely to have arisen from thedestruction of a negative element rather than the creation of a positiveelement. In addition, alteration of the candidate sequence is morelikely to destroy a positive element than create a positive element. Insome embodiments, a candidate sequence is chosen and modified so as toincrease the production of a therapeutic protein. The candidate sequencecan be modified, e.g., by sequentially altering the codons or byrandomly altering the codons in the candidate sequence. A modifiedcandidate sequence is then evaluated by determining the level ofexpression of the resulting therapeutic protein or by evaluating anotherparameter, e.g., a parameter correlated to the level of expression. Acandidate sequence which produces an increased level of a therapeuticprotein as compared to an unaltered candidate sequence is chosen.

In some embodiments, one or a group of codons can be modified, e.g.,without reference to protein or message structure and tested.Alternatively, one or more codons can be chosen on a message-levelproperty, e.g., location in a region of predetermined, e.g., high or lowGC content, location in a region having a structure such as an enhanceror silencer, location in a region that can be modified to introduce astructure such as an enhancer or silencer, location in a region having,or predicted to have, secondary or tertiary structure, e.g., intra-chainpairing, inter-chain pairing, location in a region lacking, or predictedto lack, secondary or tertiary structure, e.g., intra-chain orinter-chain pairing. A particular modified region is chosen if itproduces the desired result.

Methods which systematically generate candidate sequences are useful.For example, one or a group, e.g., a contiguous block of codons, atvarious positions of a synthetic nucleic acid sequence can be modifiedwith common codons (or with non common codons, if for example, thestarting sequence has been optimized) and the resulting sequenceevaluated. Candidates can be generated by optimizing (or de-optimizing)a given “window” of codons in the sequence to generate a firstcandidate, and then moving the window to a new position in the sequence,and optimizing (or de-optimizing) the codons in the new position underthe window to provide a second candidate. Candidates can be evaluated bydetermining the level of expression they provide, or by evaluatinganother parameter, e.g., a parameter correlated to the level ofexpression. Some parameters can be evaluated by inspection orcomputationally, e.g., the possession or lack thereof of high or low GCcontent; a sequence element such as an enhancer or silencer; secondaryor tertiary structure, e.g., intra-chain or inter-chain paring.

In some embodiments, the optimized nucleic acid sequence can express thevariant phytase polypeptide of the invention, at a level which is atleast 110%, 150%, 200%, 500%, 1,000%, 5,000% or even 10,000% of thatexpressed by nucleic acid sequence that has not been optimized

Staring with the amino acid sequence of a variant phytase, a candidateDNA sequence can be designed. During the design of the synthetic DNAsequence, the frequency of codon usage can be compared to the codonusage of the host expression organism and rare host codons can bemodified in the synthetic sequence. Additionally, the syntheticcandidate DNA sequence can be modified in order to remove undesirableenzyme restriction sites and add or alter any desired signal sequences,linkers or untranslated regions. The synthetic DNA sequence can beanalyzed for the presence of secondary structure that may interfere withthe translation process, such as G/C repeats and stem-loop structures.Before the candidate DNA sequence is synthesized, the optimized sequencedesign can be checked to verify that the sequence correctly encodes thedesired amino acid sequence. Finally, the candidate DNA sequence can besynthesized using DNA synthesis techniques, such as those known in theart.

In some embodiments, the general codon usage in a host organism, such asany of those described herein, can be utilized to optimize theexpression of the heterologous polynucleotide sequence in the hostorganism. The percentage and distribution of codons that rarely would beconsidered as preferred for a particular amino acid in the hostexpression system can be evaluated. Values of 5% and 10% usage can beused as cutoff values for the determination of rare codons.

VII. Host Cells and Production Strains

As will be appreciated by those in the art, there are a wide variety ofproduction host organisms for the recombinant production of the variantphytases of the invention, including, but not limited to bacterial cellsand fungal cells including yeast. In addition, while the G1P parentphytase is unglycoslyated, glycosylation by production in yeast andfungi does not adversely affect the phytase activity.

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant glucoamlyase of the presentinvention operably linked to one or more control sequences that directthe production of a variant of the present invention. A construct orvector comprising a polynucleotide is introduced into a host cell sothat the construct or vector is maintained as a chromosomal integrant oras a self-replicating extra-chromosomal vector as described earlier. Theterm “host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the variant and its source. In some embodiments,the host cell exhibits transitory expression of the variantglucoamlyase. In some embodiments, the host cell is a stably transfectedhost or a host cell that stably (i.e., permanently) expresses thevariant phytase. In some embodiments, the host cell is a production hostcell.

The host cell can be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote. Such host cells include butare not limited to bacterial, fungal, and yeast cells. The host cell canalso be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

The host cell can be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell can be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al, 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, BiolTechnology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

VIII. Compositions

The present invention also provides compositions comprising a variantphytases. In some embodiments, the composition comprises a carrierand/or an excipient. In some embodiments, the compositions are enrichedin such a variant phytase polypeptide of the present invention. The term“enriched” indicates that the phytase activity of the composition hasbeen increased, e.g., with an enrichment factor of at least 1. In someembodiments, the compositions are formulated to provide desirablecharacteristics such as low color, low odor and acceptable storagestability.

In some embodiments, the composition comprises a variant phytasepolypeptide of the present invention as the major enzymatic component,e.g., a mono-component composition. In some embodiments, the compositionmay comprise multiple enzymatic activities, such as an aminopeptidase,alpha-amylase, beta-amylase, phytase, isoamylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cydodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, phytase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, polyphenoloxidase,pullulanase, proteolytic enzyme, ribonuclease, transglutaminase, and/orxylanase.

IX. Methods of Production

The present invention also relates to methods of producing a variantphytase polypeptide, comprising: (a) cultivating a host cell of thepresent invention under conditions suitable for expression of thevariant phytase polypeptide; and (b) optionally recovering the variantphytase polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant phytase polypeptide using methods known in theart. For example, the cell may be cultivated by shake flask cultivation,or small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe variant to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or can be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant phytase polypeptide is secreted into thenutrient medium, the variant phytase polypeptide can be recovereddirectly from the medium. If the variant is not secreted, it can berecovered from cell lysates.

The variant phytase polypeptide can be detected using methods known inthe art that are specific for the variants. These detection methodsinclude, but are not limited to, use of specific antibodies, formationof an enzyme product, or disappearance of an enzyme substrate. Forexample, an enzyme assay may be used to determine the activity of thevariant phytase polypeptide.

The variant phytase polypeptide can be recovered using methods known inthe art. For example, the variant phytase polypeptide can be recoveredfrom the nutrient medium by conventional procedures including, but notlimited to, collection, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The variant can be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant. In a particular embodiment variant phytase of theinvention is not recovered and the host cell is a yeast host cell. Inparticular the yeast is a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell. In some embodiments, the yeastis Saccharomyces cerevisiae.

X. Phytase Formulations and Uses

As discussed herein, the use of phytase in animal feeds has a number ofbenefits, including a feed cost savings, such as reductions in dietaryinorganic phosphate, energy and amino acids, including a fast andefficient breakdown of dietary phytate and increased nutrientavailability from phytate, as well as production benefits such as bodyweight gain for the non-ruminant subjects, the increased release ofnutrients from phytate, and a significant benefit in the reducedphosphorus excretion to improve the environmental impacts ofnon-ruminant animals. In some embodiments, the variant phytases of theinvention are formulated and added to feed or can be made as a componentof the feed. In the former case, the feed stock addition of phytase canbe done by formulating the phytase on a carrier feed such as wheatflour.

As will be appreciated by those in the art, the formulation of thevariant phytases of the invention depends on its end use and theassociated conditions. Suitable formulations for the variant phytases ofthe invention include liquid formulations, dried formulations (includingspray dried formulations), powdered formulations, granular formulations,and pelleted formulations.

In some embodiments, the enzyme composition (i.e., polypeptidecompositions) of the present invention can be in any form suitable foruse, such as, for example, a crude fermentation broth with or withoutcells removed, a cell lysate with or without cellular debris, asemi-purified or purified enzyme composition, or a host cell, as asource of the enzymes.

In some embodiments, the enzyme composition can be a dry powder orgranulate, a non-dusting granulate, a liquid, a stabilized liquid, or astabilized protected enzyme. Liquid enzyme compositions may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

In some embodiments, the dosage of the polypeptide composition of theinvention and other conditions under which the composition is used maybe determined on the basis of methods known in the art.

The above compositions are suitable for use in liquefaction,saccharification, and/or fermentation processes, and in someembodiments, in starch conversion. In some embodiments, the compositionsare useful for producing a food product, including a syrup, as well asfermentation products, such as ethanol. In some embodiments, thecompositions are useful for the pharmaceutical industry, such asdigestive aids.

In one embodiment, the phytases are added to animal feed stock andpelleted as is known in the art, such that the feed is formed withphytase in it. In other embodiments, the phytase can be sprayed or dosedin a liquid form into animal feed.

EXAMPLES XI. Example 1: Gene Synthesis and Cloning

The starting gene of EcPhytase (G1P) was synthesized by GenScript(http://www.genscript.com/). The synthesized gene was cloned into thepET-20b(+) vector (Novagen EMD Millipore, USA: catalogue #69739).

XII. Example 2: Mutant Collection Design and Construction

In the first generation of improvement, a native Phytase gene (G1P, SEQID NO:1) from an E. coli strain was used as the parent. To improve thethermostability and pH tolerance of the Generation 1 parent, eightmutant collections were designed based on protein sequence andstructural analysis of EcPhytase. The design includes one to multiplespecific mutations per variant. The mutant collections were constructedusing the QuickChange® Lightning kit (Agilent Technologies, Santa Clara,Calif.) and subsequently cloned into the pET-20b(+) vector (Novagen EMDMillipore, USA: catalogue #69739).

In the second generation of improvement, the best variant from the firstgeneration was used as the parent. To further improve thethermostability and pH tolerance of the Generation 2 parent, two mutantcollections were designed based on the favorable mutations identified inthe first generation. The design includes one to multiple specificmutations per variant. The mutant collections were subsequentlyconstructed using the QuickChange® Lightning kit (Agilent Technologies,Santa Clara, Calif.).

In the third generation of improvement, the best variant from the secondgeneration was used as the parent. To further improve thethermostability and pH tolerance of the Generation 3 parent, one mutantcollection was designed based on the favorable mutations identified inthe first and second generations. The design includes one to multiplespecific mutations per variant. The mutant collections were subsequentlyconstructed using the QuickChange® Lightning kit (Agilent Technologies,Santa Clara, Calif.).

XIII. Example 3: Preparation of HTP Phytase-Containing Wet Cell Pellets

BL21(DE3)pLysS E. coli cells (Thermo Fisher Scientific, USA: Catalogue #C606003) comprising recombinant phytase-encoding genes from singlecolonies were inoculated into individual wells of 96 wells shallowmicrotiter plates holding 1800 LB containing 1% glucose and 100 μg/mLampicillin. The cultures were grown overnight at 30° C., 200 rpm and 85%humidity. 10 μL of the overnight culture from each well was transferredinto the corresponding wells of 96 deep well plates containing 390 mLTerrific Broth (TB) and 100 μg/mL ampicillin. The deep-well plates wereincubated for 3.5-4 hours (OD600 0.6-0.8) at 37° C., 250 rpm and 85%humidity. The cell cultures were then induced by IPTG to a finalconcentration of 1 mM and incubated overnight under the same conditionsas originally used. The cells were then pelleted using centrifugation at4000 rpm for 10 min at 4° C. The supernatants were discarded and thepellets frozen at −80° C. prior to lysis.

XIV. Example 4: Lysis of the HTP Phytase Plates

150 μL of B-PER bacterial protein extraction reagent (Thermo FisherScientific, USA: Catalogue #78248) was added to the cell paste in eachwell as described above. The cells were lysed at room temperature for1.5 hours with shaking on a bench top shaker. The plate was thencentrifuged for 10 min at 4000 rpm and 4° C. The clear supernatants wereused to perform biochemical assays to determine activity, pH toleranceand thermostability.

XV. Example 5: Enzymatic Assay without Temperature Treatment

The lysate from example 4 was 400 fold diluted using 0.1M sodiumacetate, pH 4.5 and pH 5.5. In 96 well shallow microtiter plates, 30 μlof the diluted lysate was added to 20 μl of sodium phytate substrate(C₆H₆Na₁₂O₂₄P₆, FW: 923.81) prepared in 0.1M sodium acetate, pH 4.5 andpH 5.5. The reaction was incubated at 24° C., 150 rpm for 30 minutes.The reaction was quenched with 50 μl of 5% w/v trichloroacetic acid. Toeach well of the 96 well shallow microtiter plates, 100 μl of coloringreagent was added. The coloring reagent was freshly prepared by mixingfour volumes of 2.5% ammonium molybdate solution in 5.5% sulfuric acidand one volume of 2.7% ferrous sulfate solution. After shaking theplates for 30 seconds, they were subjected to centrifugation at 400 μlrpm for 2 minutes. 100 μl of the supernatant from each well of thecentrifuged plates was then diluted with 100 μl of water and absorbanceread at 700 nm. The enzyme activity of variant was compared to theparent under the same conditions to determine activity improvement(FIGS. 5 and 6).

XVI. Example 6: Enzymatic Assay with Temperature Treatment

The lysate from example 4 was 90 fold diluted using 0.1M sodium acetate,pH 4.5 and pH 5.5. 50 μl of the diluted lysate was transferred to PCRplates and heated at 58° C. or 66° C. (G1), 66° C. (G2), and 70.2° C.(G3) for 5 minutes in thermocyders to identify improved variants. In 96well shallow microtiter plates, 30 μl of the treated lysate was added to20 μl of sodium phytate substrate (C₆H₆Na₁₂O₂₄P₆, FW: 923.81) preparedin 0.1M sodium acetate, pH 5.5. The reaction was incubated at 37° C.,150 rpm for 30 minutes. The reaction was quenched with 50 μl of 5% w/vtrichloroacetic acid. To each well of the 96 well shallow microtiterplates, 100 μl of coloring reagent was added. The coloring reagent wasfreshly prepared by mixing four volumes of 2.5% ammonium molybdatesolution in 5.5% sulfuric acid and one volume of 2.7% ferrous sulfatesolution. After shaking the plates for 30 seconds, they were subjectedto centrifugation at 400 μl rpm for 2 minutes. 100 μl of the supernatantfrom each well of the centrifuged plates was then diluted with 100 μl ofwater and absorbance read at 700 nm. After pH/Temperature treatment, theenzyme activity of variant was compared to the parent under the sameconditions to determine improvement in pH tolerance and thermostability.The best generation 1 variant G2P showed 12 and 20-fold improvement overthe generation 1 parent G1P at pH 4.5 and pH 5.5 respectively (FIG. 6).The best generation 2 variant G3P showed 2-fold improvement over thegeneration 2 parent G2P at pH 5.5 (FIG. 7). The best generation 3variant G4P showed 4-fold improvement over the generation 3 parent G3Pat pH 5.5 (FIG. 8).

XVII. Example 7: Validation of the Variants in Temperature GradientAssay

The top variants from each generation were selected based on improved pHtolerance and thermostability. The best variants were then subjected toa temperature gradient treatment in the range of 55° C.-75° C. or 63°C.-75° C. for 5 minutes at pH 4.5 and/or 5.5 following the protocoldescribed in example 6. FIG. 10 shows the thermostability profile ofG1P, G2P and G3P variants at pH 4.5 and 5.5 respectively. Under bothpHs, G1P maintains 100% activity till 57° C. G2P maintains 100% activitytill 64° C., whereas G3P is stable up to 68° C. FIG. 12 shows thethermostability profile of G1P, G2P, G3P and G4P variants at pH 5.5respectively.

We claim:
 1. A composition comprising a variant phytase enzymecomprising at least one amino acid substitutions as compared to SEQ IDNO:1, wherein said amino acid substitution is at a position numberselected from the group consisting of 255, 159, 354, 1, 30, 36, 39, 55,60, 65, 69, 73, 74, 79, 85, 101, 109, 111, 116, 118, 120, 137, 138, 139,141, 146, 157, 176, 180, 183, 184, 185, 186, 189, 233, 245, 276, 282,288, 291, 295, 297, 311, 315, 341, 363, 369, 370, 380, 383, 385 and 402.2. A composition comprising a variant phytase enzyme comprising at leastone amino acid substitutions as compared to SEQ ID NO:1, wherein saidamino acid substitution is at a position number selected from the groupconsisting of 255, 159, 354, 1, 30, 36, 39, 55, 60, 65, 69, 73, 74, 79,85, 101, 109, 111, 116, 118, 120, 137, 138, 139, 141, 146, 157, 176,180, 183, 184, 185, 186, 189, 233, 245, 276, 282, 288, 291, 295, 297,311, 315, 341, 363, 369, 370, 380, 383, 385 and 402, wherein saidvariant phytase enzyme has at least at least 1.1 fold better activity ascompared to SEQ ID NO:1 under a condition selected from the groupconsisting of thermostability at 58° C., thermostability at 66° C., pHstability at pH 4.5 and pH stability at pH 5.5.
 3. A compositionaccording to claim 1 wherein said variant phytase enzyme is at least 95%identical to SEQ ID NO:1.
 4. A composition according to claim 1 whereinsaid amino acid substitutions are selected from the group consisting ofY255D, R159Y, F354Y, Q1S, Q1V, Q1N, Q30K, A36K, T39D, I55V, H60S, H60Q,R65H, D69N, A73D, A73E, K74D, K74P, K74L, Q79L, Q79R, Q79A, Q79G, Q79F,I85V, A101L, A109D, A109D, A109E, A109G, A109F, A109P, T111S, T111D,T111Q, A116Y, A116P, A116R, A116S, T118R, T118S, S120R, N137S, N137P,A138V, A138H, A138D, A138P, N139P, N139A, N139H, T141E, T141G, T141A,T141R, S146R, G157Q, G157N, G157L, G157R, G157A, N176K, N180T, N180E,K183R, Q184S, D185N, D185L, E186V, E186A, S189T, G233A, Y255D, T245E,M276V, H282N, H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S,E315G, E315S, L341Y, L341V, K363A, K363L, N369P, T370P, A380R, A380T,A380P, E383S, R385S, R385V, R385T, E402R, E402T, E402D, E402P and E402N.5. A composition according to claim 1 wherein said variant phytaseenzyme has amino acid substitutions at one of said positions, two ofsaid positions, three of said positions, four of said positions, five ofsaid positions, six of said positions, seven of said positions, eight ofsaid positions, nine of said positions, ten of said positions, eleven ofsaid positions, twelve of said positions, thirteen of said positions,fourteen of said positions, fifteen of said positions, sixteen of saidpositions, seventeen of said positions, eighteen of said positions,nineteen of said positions or twenty of said positions.
 6. A compositionaccording to claim 1 wherein said variant phytase comprises the aminoacid substitutions I55V/G157Q/R159Y/Y255D/F354Y/A380P and is at least95% identical to SEQ ID NO:5.
 7. A composition according to claim 1wherein said variant phytase comprises the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P and at least one further amino acidsubstitution selected from the group consisting of Q1S, Q1V, Q1N, Q30K,A36K, T39D, H60S, H60Q, R65H, D69N, A73D, A73E, K74D, K74P, K74L, Q79L,Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F,A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S,S120R, N137S, N137P, A138V, A138H, A138D, A138P, N139P, N139A, N139H,T141E, T141G, T141A, T141R, S146R, N176K, N180T, N180E, K183R, Q184S,D185N, D185L, E186V, E186A, S189T, G233A, Y255D, T245E, M276V, H282N,H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S,L341Y, L341V, K363A, K363L, N369P, T370P, E383S, R385S, R385V, R385T,E402R, E402T, E402D, E402P and E402N.
 8. A composition according toclaim 1 wherein said variant phytase comprises the amino acidsubstitutions H60Q/D69N/K74D/S120R/N137P and at least one further aminoacid substitution selected from the group consisting of Q1S, Q1V, Q1N,Q30K, A36K, T39D, I55V, R65H, A73D, A73E, Q79L, Q79R, Q79A, Q79G, Q79F,I85V, A101L, A109D, A109D, A109E, A109G, A109F, A109P, T111S, T111D,T111Q, A116Y, A116P, A116R, A116S, T118R, T118S, A138V, A138H, A138D,A138P, N139P, N139A, N139H, T141E, T141G, T141A, T141R, S146R, G157Q,G157N, G157L, G157R, G157A, R159Y, N176K, N180T, N180E, K183R, Q184S,D185N, D185L, E186V, E186A, S189T, G233A, Y255D, T245E, Y255D, M276V,H282N, H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G,E315S, L341Y, L341V, F354Y, K363A, K363L, N369P, T370P, A380R, A380T,A380P, E383S, R385S, R385V, R385T, E402R, E402T, E402D, E402P and E402N.8. A composition according to claim 1 wherein said variant phytasecomprises the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P and atleast one further amino acid substitution selected from the groupconsisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D, R65H, A73D, A73E, Q79L,Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F,A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S,A138V, A138H, A138D, A138P, N139P, N139A, N139H, T141E, T141G, T141A,T141R, S146R, N176K, N180T, N180E, K183R, Q184S, D185N, D185L, E186V,E186A, S189T, G233A, T245E, Y255D, M276V, H282N, H282P, A288E, A288R,A288V, V291I, T295I, V297L, G311S, E315G, E315S, L341Y, L341V, K363A,K363L, N369P, T370P, E383S, R385S, R385V, R385T, E402R, E402T, E402D,E402P and E402N.
 9. A composition according to claim 1 wherein saidvariant phytase comprises the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P and is atleast 95% identical to SEQ ID NO:7.
 10. A composition according to claim1 wherein said variant phytase comprises the amino acid substitutionsN139A/N176K/D185N/E402D and at least one further amino acid substitutionselected from the group consisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D,I55V, H60S, H60Q, R65H, D69N, A73D, A73E, K74D, K74P, K74L, Q79L, Q79R,Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F, A109P,T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S, S120R,N137S, N137P, A138V, A138H, A138D, A138P, T141E, T141G, T141A, T141R,S146R, G157Q, G157N, G157L, G157R, G157A, R159Y, N180T, N180E, K183R,Q184S, E186V, E186A, S189T, G233A, Y255D, T245E, M276V, H282N, H282P,A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S, L341Y,L341V, F354Y, K363A, K363L, N369P, T370P, A380R, A380T, A380P, E383S,R385S, R385V and R385T.
 11. A composition according to claim 1 whereinsaid variant phytase comprises the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P/N139A/N176K/D185N/E402Dand at least one further amino acid substitution selected from the groupconsisting of Q1S, Q1V, Q1N, Q30K, A36K, T39D, R65H, A73D, A73E, Q79L,Q79R, Q79A, Q79G, Q79F, I85V, A101L, A109D, A109D, A109E, A109G, A109F,A109P, T111S, T111D, T111Q, A116Y, A116P, A116R, A116S, T118R, T118S,A138V, A138H, A138D, A138P, T141E, T141G, T141A, T141R, S146R, N180T,N180E, K183R, Q184S, E186V, E186A, S189T, G233A, T245E, M276V, H282N,H282P, A288E, A288R, A288V, V291I, T295I, V297L, G311S, E315G, E315S,L341Y, L341V, K363A, K363L, N369P, T370P, E383S, R385S, R385V and R385T.12. A composition according to claim 1 wherein said variant phytasecomprises the amino acid substitutionsI55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P/N139A/N176K/D185N/E402Dand is at least 95% identical to SEQ ID NO:9.
 13. A compositionaccording to claim 1 wherein said variant phytase enzyme has an aminoacid substitution set selected from the group consisting of N139H/K183R,R159Y/Y255D/V291I/V297L/G311S, I55V/Y255D/G311S/F354Y,G233A/Y255D/V291I, I85V/G157Q/V291I/V297L/G311S/F354Y, A101L/Y255D,I55V/I85V/Y255D/V291I, I55V/F354Y, I55V/I85V/Y255D/V291I/F354Y,R159Y/Y255D/V291I, A101L/R159Y/S189T/T295I/F354Y, Q30K/I85V/Y255D/A380P,G157Q/R159Y, I55V/I85V/S189T/G233A/Y255D/F354Y/A380P,I55V/I85V/S189T/V297L/G311S, I55V/I85V/A101L/G157Q/G233A/F354Y,I55V/G157Q/Y255D/V291I/V297L/F354Y, I55V/G157Q/R159Y/Y255D/F354Y/A380P,I55V/R159Y/Y255D/V297L/A380P, I55V/I85V/G157Q/G233A/Y255D/V297L/F354Y,I55V/A101L/G157Q/Y255D/V297L, I55V/A101L/G157Q/Y255D/F354Y,I55V/V291I/V297L, I55V/I85V/A101L/R159Y/S189T/Y255D/F354Y,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/D69N/T111D/N137P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/N137S/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/N137P/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/K74D/N137P/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/T111D/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137S/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/K74P/N137S/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/D69N/K74P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/K74P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/T111D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q/K74D/T111D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/D69N/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q/D69N/N137S/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D/Q157A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q/K74D/N137P/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/N137P/T141E/Q157A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R/N137P/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60Q,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137P/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/D69N/K74D/S120R/N137P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/D69N/N137P/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/K74P/T111D/S120R/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137P/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/N137P/A138V/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60S/T111D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60S/D69N/S120R/N137S/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q/N137P/A138V/T41A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/Q157L,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R/N137P,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60Q,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/S120R/N137S/A138V/Q157L,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/K74Y/S120R/A138V,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/D69N/S120R/T141A,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/K74D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/T111D,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/H60S,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/T111D/T141E/Q157N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/K74D/T111D/S120R/T141E/Q157N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T39D/K74D/S120R/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/S120R/Q157N,I55V/G157Q/R159Y/Y255D/F354Y/A380P/K74D/S120R,I55V/G157Q/R159Y/Y255D/F354Y/A380P/T111D/S120R/T141E,I55V/G157Q/R159Y/Y255D/F354Y/A380P/H60S/R65H,I55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P/N139A/N176K/D185N/E402D,I55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P/N176K/D185N/H282N/R385T,I55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P/N176K/D185N/K363A/R385T/E402T,I55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P/N139H/N176K/D185N/H282N/A288R/E315G/R385T,I55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P/N139A/N176K/A288R/E315G,andI55V/H60Q/D69N/K74D/S120R/N137P/G157Q/R159Y/Y255D/F354Y/A380P/D185N/H282N/A288R/E315G.14. A composition according to claim 1 further comprising animal feed.15. A nucleic acid encoding the phytase enzyme of claim
 1. 16. Anexpression vector comprising the nucleic acid of claim
 15. 17. A hostcell comprising the expression vector of claim
 16. 18. A method ofmaking a phytase enzyme comprising culturing the host cell of claim 17under conditions wherein said phytase enzyme is produced, and recoveringsaid enzyme.